EX-ACEA-0010MA,a Small Molecular Compound,Induces Apoptosis And Inhibits Cell Cycle Of Acute Myeloid Leukemia Cells Via PI3K/AKT Signaling Pathway

Part I Effects of EX-ACEA-0010MA treatment in acute myeloid leukemia cells.Purpose:The aim of this part is to research the effects of EX-ACEA-0010MA on cell proliferation, apoptosis, cell cycle and colony formation of acute myeloid leukemia (AML) cell linesMethods:AML Cell lines and L02cells were treated with EX-ACEA-0010MA, their viability evaluated by MTT method, the results were then compared with that of Ibrutinib. The effect of EX-ACEA-0010MA on colony formation ability of the AML cell lines was detected by colony formation assay, and that on cell cycle and apoptosis were measured by flow cytometry. Hoechst33258staining method was used morphologically to study the influence of EX-ACEA-0010MA on the apoptosis of AML cell lines.Result: (1)IC50of EX-ACEA-0010MA on AML cell lines U937and KG-1cells were1.05uM and6.48μM respectively after24hours’treatment and that after48hours were0.34μM and4.78μM, respectively. EX-ACEA-0010MA showed no obvious damaging effect on L02cells, indicating its mild side effects on normal cells. For Ibrutinib, IC50in U937, KG-1were19.46uM and44.97uM respectively for24h treatment,8.08uM and9.30uM respectively for48h treatment.(2) The side effect of EX-ACEA-0010MA on L02cells is gentler than Ibrutinib after reating L02cells with1.25μM EX-ACEA-0010MA and20μM Ibrutinib for24hours, respectively (p<0.001).(3)EX-ACEA-0010MA inhibited colony formation of U937and KG-1cells significantly and the inhibition effect is proportional to the concentration of EX-ACEA-0010MA.(3)EX-ACEA-0010MA induced apoptosis in both U937and KG-1cells.(4) EX-ACEA-0010MA at1.25μM can already arrested cell cycle of U937cells at G0/G1stage for24h treatment, while it did not influence cell cycle of KG-1cells significantly at the concentration of IC50.(5) AML cell lines after EX-ACEA-0010MA treatment showed increased DNA fragmentation and formation of apoptosis bodies in a dose dependent manner, indicating that U937and KG-1underwent cell apoptosis on EX-ACEA-0010MA treatment morphologically.Conclusion:(1) The growth-inhibition effect of EX-ACEA-0010MA on AML cell lines was stronger than that of Ibrutinib. And EX-ACEA-001OMA has no toxicity for L02cells.(2) EX-ACEA-001OMA could inhibit colony formation and induce apoptosis of AML cells significantly, also, it could arrest cell cycle of U937cells. Part Ⅱ The mechanism of EX-ACEA-0010MA treatment on acute myeloid leukemiaPurpose:The aim of this part is to explore the mechanisms by which EX-ACEA-0010MA exerts its function on AML cell lines.Methods:Total proteins from U937and KG-1cells treated by EX-ACEA-0010MA were analyzed by Western Blotting for BTK, p-BTK, PI3K/AKT signaling pathway, Caspase3, Caspase8, Caspase9, PARP, and the Bcl-2family. Total proteins were also analyzed for cell cycle dependent kinases including CDK2, CDK4and CDK6. Immunofluorescence method was used to detect the translocation of p-NF-κB (p-P65) from cytoplasm to nucleus in U937and KG-1cells after EX-ACEA-0010MA treatment.Result:(1)Western blotting assay showed that EX-ACEA-0010MA could inhibit the expression of p-BTK, whereas total BTK was unchanged.(2)As the concentration of EX-ACEA-0010MA increased, PI3K (p110) and p-AKT decreased in U937and KG-1cells, as well as the downstream protein p-IKK.(3) Western blotting assay also showed that Caspase3, Caspase8, but not Caspase9, and PARP were all activated.(4) Expression of p-P65and its translocation were significantly inhibited after lOuM EX-ACEA-0010MA treatment, observed with the method of immumofluorescence.Conclusion:(1) EX-ACEA-0010MA can down-regulate PI3K/AKT pathway to inhibit the phosphorylation of P65which prevente it from entering the nucleus to act as a transcription factor, thus induce apoptosis.(2) EX-ACEA-0010MA decreased the expression of cycle dependent kinase2(CDK2), CDK4and CKD6to prevent cells from entering S phase and inhibit the replication of DNA in leukemia cells.(3) The apoptosis-inducing effect of EX-ACEA-0010MA in AML cells was partially dependent on the activation of Caspase proteins, mainly the exogenous apoptosis pathway.